1. Field of the Invention
The invention is directed to the field of rapid prototyping, testing, characterizing and archiving performance parameters of microbiochips using actual body fluids.
2. Description of the Prior Art
In all fields of engineering practice, designers rely on common tools to efficiently design and produce functional working pieces. These tools include standardized manufacturing techniques, fully developed design rules and principles, databases of material properties, and visualization tools. In the past 30 years, most of these tools have been integrated into one delivery system, namely the computer. Computer aided design, in the form of modeling and simulation, is the predominant mode for performing complex engineering design.
Unfortunately for biofluidic technologies, these tools are in their infancy. Although the fundamental physics of small-scale fluidic flow is understood, the physical phenomena which occur due to surface effects, electronic effects, chemical effects, non-homogeneous media, and other deviations from a perfect system are not well known or understood. Several simulation packages exist which claim to accurately model microfluidic systems. However, given the limited number of complex microfluidic systems which have been developed and the corresponding lack of data thus far, it is unlikely that current packages will adequately serve a future industry based on complex integrated biofluidic systems. The most likely scenario is that simulation packages will evolve with the industry as more devices are built, more materials and designs are attempted, and more data is collected.
Of particular interest is the nature of biological fluid flow, where we can expect the complex behavior to be very pronounced. Biological fluids, such as blood and lipid-rich fluids are highly non-ideal, nonhomogeneous, and may be reactive to the surfaces within a microfluidic system. Fluid particulates (e.g., cells) may aggregate and affect the flow through components in ways which are not well modeled with ideal fluids such as water. Furthermore, the internal dynamics of soft, non-spherical particles in the system will dramatically alter the nature of the fluid flow. We can expect the existence of internal fluidic constituents to strongly affect the behavior of fluids in systems with low Reynolds number, where internal forces dominate and inertial effects are negligible.
Heretofore, there is very little data on the nature of non-ideal fluid flow in complex microfluidic systems, particularly systems likely to be used for biofluidic chips. Data that exists is typically suited only to the specific invention of interest, and not easily compared between one study to the next. Few, if any, studies exist which present quantitative information about the flow characteristics in fluidic systems where key design variables have been systematically varied. No doubt this is due to the relative difficulty in producing many custom microfluidic chips as well as the difficulty in obtaining detailed, high resolution flow data from within such chips.
Most measurements in microfluidic devices have been limited to bulk properties of ideal fluid flow, such as wall pressure, bulk velocity, and specific impulse. Moreover, most measurements have been for simple systems (such as capillaries), or as part of a performance characterization for a device. Reports of full three dimensional mapping of velocity fields are rare, especially for microfluidic devices.
In cases where velocity fields have been measured, the measurements have been limited to ideal fluids in simple channels primarily using particle image tracing techniques. In practice, image streaks are difficult to analyze numerically. Velocity measurements determined from particle streaks are less reliable and are about ten times less accurate than pulsed velocimetry measurements. Many published results have been made to demonstrate a particular method or measurement, rather than as an attempt to perform a thorough study of flow characteristics.
Work in optical Doppler tomography, ODT, has been reported by other groups, but the efforts have been focused mainly on characterizing flow in animal tissues. Moreover, current ODT (sometimes referred to as CDOCT, color Doppler optical coherence tomography) efforts demonstrate image resolution which is on the order of 20 μm, with scan times measured in minutes. This quality is not sufficiently good to be adapted to the needs of microfluidic chip characterization.
Blood flow in capillary systems continues to be the subject of extensive in vivo and in vitro clinical and physiological research. However, it is widely believed that experimental work has been constrained by the limitations of currently available techniques capable of resolving cellular flow behavior. Blood is a complex fluid consisting of a liquid phase, the plasma, with included bodies, the cells. The effective viscosity of whole blood is a non-linear function of flow rate. At low flow rates the viscosity is dominated by erythrocyte (red cell) aggregation. At higher flow rates, deformation of the soft erythrocytes under flow cause a change in viscosity. The large fraction of blood represented by the cells (40%-50% by volume) and their intrinsic ability to radically deform under flow conditions make this a major non-linear effect.
The erythrocyte's morphology, an underfilled, supple, membrane sack enclosing a non-nucleated fluid stroma, facilitates the erythrocyte's deformation from their static, 8 μm diameter biconcave discoid shape, to a streamlined, flow-oriented, form at high fluid shear rates (>50/sec). This results in a reduction of their flow resistance and consequently a reduction in the viscosity of the whole blood.
Ideal fluids lend little to the understanding of blood flow in microscopic systems. Within microscopic channels and orifices, individual cells must mechanically deform to pass serially through passages. Rigid cells or contaminant particles can cause clogging and blocking within microscopic components, and most certainly modify the intended behavior of the components. Even steady state flow in blood is a complicated state, consisting of an interplay between the restoring forces exerted by the deformed cells, the fluid flow of a thin lubricating layer between the cell and channel walls, and biosurface interactions between the cell membrane and the channel wall. Blood flow in the microscopic regime cannot be treated as that of an ideal or homogeneous liquid.
Given that most data are for simple systems (such as channels), for ideal fluids (such as water), and of only bulk properties (such as flow vs. pressure), there is a need for a tool with which actual blood flow can be studied. Such data are of the most relevant to the design and integration of complex microfluidic systems.
Most measurements in microfluidic devices have been limited to bulk properties of ideal fluid flow, such as friction factors, wall pressure, bulk velocity, and specific impulse. Moreover, most measurements have been for simple systems (such as capillaries), or as part of a performance characterization for a specific, novel invention.
In cases where velocity fields have been measured, the measurements have been limited to ideal fluids in simple channels primarily using particle image tracing techniques. In practice, image streaks are difficult to analyze numerically. Velocity measurements determined from particle streaks are less reliable and are about ten times less accurate than pulsed velocimetry measurements. Many published results have been made to demonstrate a particular method, rather than as an attempt to perform a thorough study of flow characteristics.
Typical are the following examples. Brody et al. “Biotechnology at Low Reynolds Numbers,” Biophys J 71: 3430 D3441 (1996) used an intensified CCD camera to image particle streaks in an 11 μm by 72 μm (cross section) silicon channel. Also, lanzilloto et al. “A Study of Structure and Motion in Fluidic Microsystems,” AIAA (1997) used X-ray micro-imaging techniques to measure velocity fields in 500-1000 μm diameter micro-tubes by recording the motion of 1-20 μm emulsion droplets in a liquid flow. Velocity fields were estimated by tracking the trajectories of the emulsion droplets over time. They reported mean velocity fields in an 840 μm diameter tube, with vector-vector spacings of about 40 μm and axial bulk velocities of 7-14 μm/s.
A high resolution system for imaging particle traces has also been demonstrated. The results were for a two dimensional flow of water between two glass slides and served mostly to demonstrate the capability of a particular device.
A two-laser fluorescence-based imaging technique was recently demonstrated Arnold et. al., “Fluorescence based Visualization of Electroosmotic Flow in Microfabricated Systems,” SPIE Conference on Microfluidic Devices and Systems II, Vo. 3877 (1999). This technique used a fluorescent tracer which was created by photoactivation of a caged fluorescein dye. The dye was uncaged using the first laser to the effect of optically ‘injecting’ a narrow band of fluorescent material into the flow channel. A second laser was used to excite the uncaged dye molecules for mapping the flow image. Like other imaging-based methods, this technique suffers from a difficulty in determining quantitative data for velocity fields, and is limited two dimensional imaging in optically transparent media.
A study by Bousse et al “Optimization of Sample Injection Components in Electrokinetic Microfluidic Systems”, IEEE Press (1999) studied the electro-osmotic flow within a capillary electrophoresis chip, and compared the results to calculations from a microfluidic simulation package (Microcosm's FlumeCAD). A series of experiments were performed where voltages were varied and subsequent flow observed. Images were qualitatively compared with modeling results, and the bulk flow velocity was measured. The results served to verify the quality of modeling. The authors did not perform a systematic study of effects of different channel designs and geometries, nor attempt to determine detailed flow profiles.
Currently, there is very little data on the nature of non-ideal fluid flow in complex microfluidic systems, particularly systems likely to be used for biofluidic chips. Data that exists is typically suited only to the specific application of interest, and not easily compared between one study to the next. Few, if any, studies exist which present quantitative information about the flow characteristics in fluidic systems where key design variables have been systematically varied. No doubt this is due to the relative difficulty in producing many custom microfluidic chips as well as the difficulty in obtaining detailed, high resolution flow data from within such chips.
A great deal of work has been done in two-dimensional array bio-fluidic systems implemented in silicon, glass, ceramic and plastics to investigate fluidic chip fabrication methods, study the flow behavior of ideal fluids, and to integrate the fluidic chip with electronics. However, only limited information is available dealing with the non-ideal inhomogeneous biological fluids such as blood. Furthermore, very few have reported constructing and characterizing three-dimensional (laminated) Bio-Flips, investigating the flow behavior in each layer, and integrating various active components. As the two-dimensional Bio-Flips technology matures for serial processing of samples, complex three-dimensional Bio-Flips technology will be the next wave for large scale parallel processing of samples and more sophisticated analysis chips with higher functionality.
For complex three dimensional biofluidic chips to be a manufacturable technology, they must allow for testing and evaluation during and after the production process. Acceptable production yield and quality control require early detection of flaws or processing problems. Within the microelectronics industry, for example, test circuits are built into the wafer for wafer-level testing. During packaging, the devices themselves are often tested to ensure high quality products, and to satisfy many design specifications, such as those required for military uses (e.g., MIL specs). Often testing is done on a representative sample under extreme conditions to satisfy quality criteria.
Unlike microelectronic devices, microfluidic devices cannot be easily probed. While a simple contact from an external probing conductor to a conducting trace allows one to make an electrical measurement without destroying the device, an analogous measurement for a microfluidic channel requires one to puncture the microchannel, thus destroying it in the process.
Therefore, what is needed instead is a non-invasive technique to allow a similar test functionality within microfluidic chips using actual body fluids.